The production, interconversion, and transfer of C1 units is an important metabolic system in all of biology. Methylotrophs are microorganisms capable of growth on C1 compounds as sole carbon and energy sources, and methylotrophy can be viewed as a specialized version of the C1 metabolism found in all organisms. A distinguishing feature of methylotrophic metabolism is the generation and consumption of formaldehyde as a central intermediate, the starting point for all of metabolism. We have gained major new insights into the pathways that consume formaldehyde in Methylobacterium extorquens AM1, and have developed a model for how the cell controls formaldehyde flux to achieve a dynamic balance of carbon and energy metabolism, avoiding formaldehyde toxicity. In this project, we will focus on the three pathways, or modules, that our working model predict are central to understanding flux of formaldehyde and energy metabolism, the H4MPT pathway, the H4F pathway, and the 3 formate dehydrogenases. We propose to begin to test our conceptual model of formaldehyde-related central metabolism in methylotrophy using a combination of biochemical, genetic, genomic, and computational approaches, focused initially on understanding how the cell responds to changes in the formaldehyde production rate. This complex system has two fundamental circuits that will be analyzed, the genetic circuit, consisting of the transcriptional and translational elements and the associated signaling components, with the output being transcripts and proteins, and the metabolic circuit, consisting of enzymes, cofactors, intermediates, and the associated signaling compounds, with the output being metabolic flux. Because of the difficulty of measuring all of the components and their characteristics, we will take a modular approach and measure outputs for each of the modules, integrating the results to create a systems-level understanding of response and resultant effects. The specific aims are: 1. Analyze the output of the genetic circuit with microarrays and proteomics. 2. Analyze the output of the metabolic circuit with enzyme assays, metabolite measurements, and direct flux measurements. 3. Integrate the results using computational models that correlate the functioning of the genetic circuit and the metabolic circuit. The result of this study will be a systems-level understanding of formaldehyde metabolism in methylotrophy. These approaches will provide a model for functional genomics at the physiological level, and will create a platform for future studies of the interaction between normal and stressed metabolism, and the mechanistic understanding of the interplay between genetic and metabolic circuits.